Solid phase immunoassays involve the immobilization of biomolecules, primarily proteins, to a surface (e.g., polystyrene, nitrocellulose) via passive or covalent interactions. However, non-specific binding (NSB) of other proteins or biomolecules to non-occupied spaces on the surface during the subsequent steps of the assay is detrimental to the specificity and sensitivity of the assay results. This NSB can be minimized by saturating these non-occupied sites with a blocking reagent, a collective term used to mean various substances that are used to reduce NSB and have no active part in the immunochemical reactions of the assay. Another function of blocking reagents is to stabilize the specifically bound biomolecules and prevent denaturation that can result in loss of immunological or enzymatic activity.
The two classes of blockers now in use for immunoassay are detergents and proteins (Batteiger, 1988).
Non-ionic detergents inhibit non-specific hydrophobic binding to surfaces; they are considered non-permanent blockers since they do not attach to the surface and their effectiveness can be nullified by washing the surface with water or buffers. Non-ionic detergent blockers are typically used only in conjunction with protein blockers.
Proteins are a class of blocking reagents that inhibit both non-specific protein-surface interactions, and protein-protein interactions. They are considered permanent blockers since they attach to the surface. Typical protein blockers include whole normal, pre-immunization mammalian serum, bovine serum albumin (BSA), non-fat dry milk, and fish gelatin (collagen).
Immunological blockers are usually taken from mammalian species such as cattle or goats for most mammalian immunoassays. Prior use of fish products as blockers and stabilizers has been confined to fish gelatin (Norland, 1986). This is derived or processed from collagen, a single protein found in the skin of the carcasses of codfish and other species, usually taken from fish processing plants. Collagen is a structural protein, and is not a component of fish serum.
All of the above blockers have disadvantages that lead to assay problems such as false negative and false positive test results. These factors are summarized below in Table 1.
TABLE 1 ______________________________________ Advantages and Disadvantages of Major Blocking Reagents Reagent Advantage Disadvantage ______________________________________ Non-fat dry milk Inexpensive Deteriorates rapidly Low Covalent NSB Quality varies Protein A Masks some antigens compatible Cross-reacts with Low cross- anti-DNA antibodies reactivity BSA Inexpensive May not be fatty-acid Low protein- free surface NSB Quality varies Well documented Single protein/high Protein A covalent NSB compatible Cross-reacts with antibodies to BSA-haptens Fish gelatin Inexpensive Severely masks Low-protein- antigens protein NSB Quality varies greatly Protein A Inadequate when used compatible alone Mammalian serum Low protein- Expensive surface NSB Incompatible with Low covalent NSB Protein A Low protein- Cross-reacts with anti- protein NSB IgG antibody Stabilizes protein ______________________________________
As shown, the use of mammalian sera or sera components as a blocking reagent can cause cross-reactions in immunoassays that employ other mammalian-derived reagents. The use of fish serum, because of its non-mammalian origin, limits cross-species reactivity.
Although the qualities needed in a blocker may differ somewhat for different applications, the ideal blocker would:
1. Inhibit non-specific binding (NSB) of assay components to the surface including non-specific hydrophobic, ionic, and covalent binding;
2. Inhibit non-specific protein-protein interactions;
3. Exhibit no cross-reactivity with assay components, especially antibodies and Protein A;
4. Minimize effects of protein denaturation that occur with phase transitions associated with immobilization and/or drying;
5. Act as a stabilizer for proteins when used in the diluent of reagents that are stored refrigerated or frozen;
6. Exhibit low enzyme activity (i.e. peroxidase, alkaline phosphatase); and
7. Not disrupt the bonds that immobilize the specific protein or biomolecule.
In addition, the ideal blocker must be free of infectious agents, and must have consistent, reproducible performance.
Because of the wide phylogenetic differences between fishes and mammals, fish serum would seem an unlikely substitute for mammalian serum to those skilled in the art. Indeed, a review of the literature on mammalian immunoassay shows no attempt to employ fish serum as a blocker. Thus, although serum from cultured fishes exhibits many of the qualities of the ideal blocker, any method for using fish serum to stabilize immunologically active reagents, and block nonspecific reactions in mammalian immunoassays remains novel. Fish serum has also not been used previously because uniform populations of fish and methods for production of large pools of high quality serum from these fish have not been available.